Assessment of artificial miRNA architectures for higher knockdown efficiencies without the undesired effects in mice

Hiromi Miura, Hidetoshi Inoko, Masafumi Tanaka, Hirofumi Nakaoka, Minoru Kimura, Channabasavaiah B Gurumurthy, Masahiro Sato, Masato Ohtsuka

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both "-MG amiRNA" cells and "amiRNA low-expression" cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs.

Original languageEnglish (US)
Article numbere0135919
JournalPloS one
Volume10
Issue number8
DOIs
StatePublished - Aug 18 2015

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MicroRNAs
microRNA
mice
Gene Transfer Techniques
Genes
Hemizygote
injection
Artificial Cells
Injections
genetic markers
cataract
Homozygote
Gene Silencing
cells
transfection
RNA Interference
homozygosity
Cataract
growth retardation
Transfection

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Assessment of artificial miRNA architectures for higher knockdown efficiencies without the undesired effects in mice. / Miura, Hiromi; Inoko, Hidetoshi; Tanaka, Masafumi; Nakaoka, Hirofumi; Kimura, Minoru; Gurumurthy, Channabasavaiah B; Sato, Masahiro; Ohtsuka, Masato.

In: PloS one, Vol. 10, No. 8, e0135919, 18.08.2015.

Research output: Contribution to journalArticle

Miura, Hiromi ; Inoko, Hidetoshi ; Tanaka, Masafumi ; Nakaoka, Hirofumi ; Kimura, Minoru ; Gurumurthy, Channabasavaiah B ; Sato, Masahiro ; Ohtsuka, Masato. / Assessment of artificial miRNA architectures for higher knockdown efficiencies without the undesired effects in mice. In: PloS one. 2015 ; Vol. 10, No. 8.
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